Rapid competitive PCR determination of relative gene expression in ...
ClinicalChemistry 42:2 227-23
1
Molecular Pathology
(1996)
and Genetks
Rapid competitive PCR determination of relative gene expression in limiting tissue samples YL-HAL
JIANG,
LAURIE
A.
DAVIDSON,
JOANNE
Reverse transcriptase (R1)-PCR is widely used to study gene transcription in many biological systems. Despite the development of a variety of procedures, quantification of RT-PCR products remains difficult, particularly when pro-
sis, agarose
TERMS:
reverse
#{149} protein
transcriptase
kinase
#{149} mRNA.
and
LUPTON,
ROBERT
S.
CHAPKLN*
series of sensitive applications for quantification of specific mRNAs in biological samples [2-4J. Quantitative PCR is rapidly becoming a common procedure because of its high sensitivity and relative speed compared with Northern blot hybridization or RNase protection assays /1]. Most PCR applications for examining gene expression can be divided into two categories, competitive and noncompetitive. Competitive reverse transcriptase (RT)-PCR, the most widely used and well-established method, involves same-tube coamplification of the gene of interest and a synthetic internal standard fragment [3]I It is necessary, in competitive RT-PCR, to perform a series of amplifications with different amounts of target and competitor. Therefore, it is not suited for routine use, especially for processing a large number of tissue samples, since it requires several reaction tubes for each unknown sample, increasing the requirement for total RNA as well as time and reagents. In noncompetitive RT-PCR, it is necessary to normalize with an internal reference gene, such as actin or glyceraldehyde-3-phosphate dehydrogenase. Therefore, a second competitive PCR must be performed for each sample [2, 4]. Alternatively, quantification of PCR products by isotope incorporation or Southern hybridization with radioactive probes is inconvenient. In all, these tedious methods are useful for processing a small number of samples. In our studies on the relation between gene expression of protein kinase C (PKC) isoenzymes and colon tumorigenesis, we developed the more rapid reverse competitive (RC)-PCR approach for the analysis of a large number of very limiting colonic mucosal specimens. This strategy represents a modification of the competitive RT-PCR approach. Basically, relative gene expression is determined by coamplification of an exogenous DNA fragment of a different size with respect to the sample cDNA, but with identical 5’- and 3’-end sequences, so that both products are amplified with the same primers. After PCR, the amplified DNA was resolved by gel electrophoresis. The relative expression of specific genes is determined by densitometric analysis of both target and competitor hands. A major advantage of the RC-PCR procedure is that only one tube
cessing a large number of samples. Therefore, we developed a novel alternative PCR technique that we term “rapid competitive PCR” (RC-PCR), designed to study the relative expression of specific genes in a large number of small tissue biopsies. RC-PCR is characterized by measuring relative gene expression at the mRNA level of two or more samples with a nonradioactive assay based on competitive PCR amplification between identical sequences of internal standard and target cDNA. Only a single reaction tube per sample is used in this technique, and it was validated by comparing RC-PCR of protein kinase C and a expression in rat colonic mucosa samples with competitive RT-PCR analysis (requiring 6-8 reaction tubes per sample). We conclude that RC-PCR is a simple, rapid, highly sensitive technique that is capable of detecting less than twofold differences in mIRNA expression. INDEXING
R.
electrophore-
C
The quantitative measurement of gene expression is of major importance in addressing many fundamental questions in biology and medicine. However, in many clinical and animal studies, determination of the relative expression of specific mRNAs in a large number of small tissue samples is required to assess gene expression under physiological and pathological conditions. Northern analysis and RNase protection assays, widely used to quantify specific mRNAs, require large amounts of total RNA and are time consuming. Thus, they are not suitable for large clinical and animal studies requiring gene expression analysis [1]. The advent of PCR has resulted in the implementation of a
Faculty of Nutntion. Molecular and Cell Biology Group, l’exas A&M University, College Station, TX 77843-247!. * Address correspondence to this author at: 442 Kleherg Center, ‘Texas A&M University, College Station, TX 77843-247!. Fax 409-845-6433; e-mail [email protected]. Received September I, 1995; accepted October 30, 1995.
Nonstandard rapid competitive
227
abbreviations: PCR;
and PKC.
R1’-PCR, protein
reverse kinase
transcriptase C.
PCR;
RC-PCR,
et al.: Rapid competitive
Jiang
228
PCR
of mRNA
____
Ja
Primers
cDNA
+
1#{176}PCR
-s.b
Generation
of
Va
Composite primer
‘%
internal
C
standard
__________________ Competitor (internal
I
I
standard, 584 bp)
b
__,__.._.*.*#iInternal
B
CDNA
cI)NA, equal amounts
IS
Serial dilutions of IS
4,
RC-PCR
PCR and Electrophoresis
and RT.PCR
680 bp
PCR and Electrophoresis
680 584
bp
Competitive
RC-PCR
Fig. 1. Schematic
view of internal standard
generation
with subsequent
RC-PCR and competitive
RT-PCR
RT-PCR.
(A) Generation of internal standard. The black regions on the cDNA are complementary
to PcR primers b and c. To produce a smaller intemal standard (competitor), primer a contains primer cat its 5 end and a 13-bp region at its 3 end, which is designed PCR, primers b and c are used to generate a large quantity of the internal standard. (B)
primer band composite primer a were used during 1* PCR. The composite complementary region of the eDNA. During 2* Quantification of relative PKC gene expression by using RC-PCR and competitive RT.PCR.For RC-PCR,one tube containing cDNA and internal standard (IS) is amplified, electrophoresed. stained, and scanned for quantification. For competitive RT-PcR, seven tubes containing equal amounts of eDNA and dilutions of internal standard are amplified, electrophoresed, stained, and the equivalence estimated by eye. 680 bp: targeted PKC gene; 584 bp: internal standard (competitor). to anneal to an internal
is required for coamplification of the gene of interest and internal standard. For the purpose of validation of RC-PCR methodology, competitive RT-PCR was performed simultaneously to measure the relative expression of PKC and a mRNA in eight different rat colonic mucosal samples. Both methodologies gave similar results.
Materials and Methods RNA
EXTRACTION
AND
REVERSE
TRANSCRIPTION
Total RNA was extracted from rat colonic mucosa by using Totally RNA’ as per the manufacturer’s instructions (Ambion, Austin, TX). First-strand cDNA was synthesized in a 20-/.LL total volume by using oligo(dT) primer and SuperScript II reverse transcriptase (Gibco BRL, Gaithersburg, MD) as we have previously described [5].Briefly, rat colon total RNA (1 j.Lg) and 300 ng of oligo(dT) primer were denatured by heating to 70 #{176}C for 10 mm, quickly chilled on ice, and subsequently reverse-transcribed by incubation with 0.01 mol/L dithiothreitol, 1 mmol/L dNTPs, and 200 U of SuperScript II reverse transcriptase for 1 h at 37 #{176}C, followed by enzyme denaturation at 95 #{176}C for 5 mm. Incubations containing no reverse transcriptase were used as negative controls. COMPETITIVE
RT-PCR
For validation purposes, competitive RT-PCR was performed. A schematic view of internal standard generation is shown in
Fig. 1. A single set of primers to amplify both target cDNA and an added competitor (internal standard) of known concentration was used. Internal standards for PKC and a were constructed as previously described [6, 7]. The primer pairs used for amplification of PKC and a are listed in Table 1. For each sample, 1 jg of total RNA was routinely used for cDNA synthesis. PCR was conducted with a GeneAmp PCR System 2400 (PerkinElmer, Foster City, CA) in a total volume of 50 .tL containing 1 .tL (0-100 ng) of cDNA template and 1 .tL of internal standard (156.2 5-10 000 fg), 20 pmol of each primer, 500
Table 1. PrImer pairs
for constructionof internal standards of PKC Cand a mRNA.
and amplification
PKC competitor (584 bp) Forward: 5’-CGATGGGGTGGATGGGATCAAAA-3’ Reverse: 5’-GTATrCATGTCAGGI3TTGTCTGGAmCGGGGGCG-3’ PKC mRNA (680 bp) Forward: 5’-CGATGGGGTGGATGGGATCAAAA-3’ Reverse: 5’-GTAUCATGTCAGGG11GTCTG-3’ PKC a competitor (293 bp) Forward: 5’-TGAACCCTCAGTGGA.ATGAGT-3’ Reverse: 5’-GGCTGCTrCCTGTCTTCTGAACTTGGCmCTCGAAC-3’ PKC a mRNA (325 bp) Forward: 5’-TGAACCCTCAGTGGAATGAGT-3’ Reverse: 5-GGCTGC1TCCTGTCTrCTGAA-3’
Clinical Chemistiy 42, No. 2, 1996
jimollL dNTPs, 10 mmol/L Tris-HCI (pH 9.0), 50 mmol/L KC1, 1.5 mmol/L MgCI,, and 1.25 U of Taq DNA polymerase (Gibco BRL). To minimize tube-to-tube differences, a master amplification mixture was prepared for every assay. An amplification program (28 cycles) of denaniration (93 #{176}C, 15 s), annealing (59 #{176}C, 15 s), and extension (74 #{176}C, 45 s) was used, followed by incubation at 74 #{176}C for 7 mm. All reactions were initially denatured at 93 #{176}C for 1 mm before PCR amplification. After completion of PCR, 10-g.tL aliquots of the reaction mixtures were analyzed on 4% NuSieve agarose gels (FMC Bioproducts, Rockland, ME), followed by staining in ethidium bromide. Sample mRNA was quantified by titrating a known quantity of the internal standard against a constant amount of the sample cDNA target. The concentration at which the product from the standard target equalled the product from the experimental target was taken to be the concentration of the sample mRNA. The relative amount of sample (target) mRNA was calculated by assigning an arbitrary sample a value of 1 and establishing a relative index to allow for comparison with RC-PCR results described below. RELATIVE
EXPRESSION
OF SPECIFIC
MRNA
MEASURED
BY
RC-PCR
RC-PCR, reverse transcription and PCR were performed as described above, with the following modifications. For each sample, only one PCR tube was used instead of seven tubes in competitive RT-PCR (Fig. 1). All samples were supplemented with an identical amount of competitor, and PCR products were For
separated
gel electrophoresis. A range of RNA numbers, and the ratio between internal standard and sample were determined to ensure that amplification was quantitative. Band intensities of photographed ethidium bromide-stained gels were determined with a BioImage Visage 1 lO-2D imaging instrument (Kodak, Ann Arbor, MI) to quantify colonic PKC and a gene expression. The relative amount of sample mRNA was calculated by assigning an arbitrary sample a value of 1 and establishing a relative index. different
OF INTERNAL
To eliminate during DNA used
as
STANDARDS
the confounding effect of variable PCR efficiency amplification, we generated DNA fragments that internal standards for both competitive PCR and
RC-PCR. The procedure used to produce internal standard DNA fragments was described previously [6, 7]. Briefly, a composite primer listed in Table 1 was used in PCR reactions to generate a slightly smaller product, which was confirmed by restriction enzyme digestion. This DNA fragment was subsequently purified, quantified by ultraviolet absorption, and used in both competitive RT-PCR and RC-PCR for the measurement of the relative expression of PKC and a rnRNAs in colonic mucosa. RC-PCR
CONDITIONS
establish
expression optimal
I-
0 Si
C.
E 0 0
I-
C is
is C ISi
C
Si I-
is
I-
0
40
20
60
80
100
120
RNA (ng) Fig. 2. (A) Electrophoresis of products from rat colon PKC RC-PCR; (B) linear plot of data obtained by densitometric scanning of (A). (A) Total RNA(1 sag)was reverse-transcribed into cDNA as described in Materials and Methods, and twofold dilutions of rat colonic total RNA (lanes, from left to right:0, 3.125, 6.25, 12.5, 25, 50, 100 ng) were mixed with a constant amount (1 pg) of internal standard DNA (PKC competitor). PCR products were separated with a 4% NuSieve agarose gel, followed by ethidium bromide (5 mg/L) staining. PRC, rat PKC pcR product band (680 bp); IS, internal standard (584 bp). (B) The ratio (target/competitor) from densitometry of the gel shown in (A) was plotted against the amount of cDNA.Regression line slope = 0.08 (R2 = 0.96).
cycle
Results PREPARATION
To
A
by agarose
amounts,
were
229
of
PCR
AND
ASSAY
RC-PCR
for
specific
genes,
conditions.
the Once
it
LIMITS
measurement of the relative was necessary to determine the reaction conditions were
established and optilnized, the appropriate starting amount of sample RNA was empirically determined to ensure that a constant ratio between the target and the internal standard existed. For the purpose of determining the effect of sample mass (initial PCR reaction template) on the ratio of amplified sample to internal standard, RNA (1 ig) was reverse-transcribed to generate cDNA. Subsequently, a series of twofold dilutions of the cDNA (equivalent to 1.5625-100 ng of starting RNA) was coamplifled with a constant amount of PKC internal standard at the optimal number of cycles (28 cycles) to determine the linear reaction range (Fig. 2). The linear amplification range for PKC and a was from 10-100 ng and 5-50 ng of sample cDNA, respectively. To examine the effect of cycle number on PKC mRNA expression in rat colon, cDNA from 40 ng of total RNA and a constant amount of internal standard were coamnplified by RC-PCR over a range of amplification cycles. The proportionality of both target and standard mass remained constant from 21 to 32 cycles (Fig. 3). COMPARISON
To validate
OF RC-PCR
RC-PCR
AND
COMPETITIVE
methodology,
RT-PCR
we compared
results
(rela-
tive gene expression) with those obtained by competitive RTPCR. Total RNA preparations from rat colon were subjected to competitive RT-PCR with increasing amounts of internal standard (competitor DNA) (Fig. 4), and the absolute amount and relative expression of mRNA were calculated (Table 2). The relative expression of PKC and PKC a in colonic samples
Jiang
230
et al.: Rapid
competitive
PCR
of mRNA
total RNA samples were divided into eight aliquots each and individually reverse-transcribed. Subsequently, four aliquots of each reverse transcriptase reaction were utilized for both PKC ,T and a RC-PCR assays. The results of the assays are shown in Table 3. The standard error and covariance (CV) were consistently low, indicating that the reverse transcriptase and PCR components of the assay are highly reproducible.
A
1.5
Discussion 19
21
23
25
27
29
31
33
35
Cycle Number
Fig. 3.
(A) RC-PCR cycle titration of colonic PKC ; (B)plot of data obtained by densitometric scanning of (A). (A) Total RNA(1 extracted from rat colonic mucosa was reverse-transcribed. Equal amounts subsequently
of cDNA from an aliquot corresponding to 20 ng of total RNA was amplified in the presence of a constant amount of internal standard (1 pg) for 19-34 cycles (lanes, from left to right: 19, 21, 24. 26, 28, 30. 32. 34 cycles). PCR products (10 L) were separated with a 4% NuSieve agarose gel, followed by ethidium bromide (5 mg/L) staining. PK , rat PKC PCRproduct band (680 bp); IS, internal standard (PKC competitor, 584 bp). (B) The ratio (target/competitor) from densitometry of gel shown in (A) was plotted
against cycle number. determined by RC-PCR was very similar to competitive RTPCR data, as shown in Table 2. To evaluate the reproducibility of the assay and variability of the reverse transcription step, three different rat colonic mucosa
I......Is
c-I
C Si C.
E 0
c-I is
C
is Si
C
0
2000
4000
6000
fg Competitor
8000
10000
12000
DNA
Fig. 4. (A) Competitive RT-PCR of colonic PKC ; (B) linear plot of data obtained by densitometric scanning of (A). (A) Total RNA(1 p.g)extracted from rat colonic mucosa was reverse-transcribed and amplified in the presence of different amounts of competitor DNA (lanes, from
left to right: 10 000,
5000, 2500, 1250, 625, 312.5, 156.25 fg). PcR (10 L) were separated with a 4% NuSieve agarose gel, followed by ethidium bromide (5 mg/L) staining. PKc , rat PKc PCR product band (680 bp); IS, internal standard (584 bp). (B) The ratio of the internal standard to the sample allows for the quantification of PKC mRNA. At the theoretical equivalence point (ratio = 1), the amount of PKC competitor DNA (internal standard) equals the amount of PKC cDNA present in the analyzed sample.
products
We report the development and validation of a simple, nonradioactive approach (RC-PCR) to determine the relative expression of specific genes. This rapid and highly reproducible technique exploits the tremendous sensitivity of competitive PCR, and has several advantages over competitive RT-PCR: Numnerous samples can be processed by RC-PCR in the same run; less total RNA is required; and the technique is much less labor intensive, costly, and time-consuming. With conventional PCR, small variations in the starting concentration of the target sequence and the efficiency of Taq DNA polymerase at each cycle can result in very large variations in the final yield of PCR product [8, 9]. Thus, quantitative comparisons between tubes are invalid because the amount of final product does not accurately reflect the amount of starting template. This is particularly true when the number of cycles is excessive and amplification efficiency decreases dramatically (plateau effect). To address these pitfalls, several approaches have been developed for quantitative RT-PCR, including (a) cycle titration, (b) serial dilutions, (c) coamplification of an unrelated endogenous mRNA for normalization of the signal, and (d) competitive PCR. The latter method [8, 10, 11] is the most rigorous and is accomplished by introducing into the sample a known amount of an exogeneous, mutated artificial internal standard that may be coamplifled in the same tube with the same primers as the mRNA of interest. The two products that are amplified may be distinguished from one another by size, restriction endonuclease cleavage, or hybridization with separate probes. Since the internal standard is coamplified with the same primers as the target sequence, the efficiency of amplification is the same as that for the endogenous mRNA. Tube-to-tube variation is thereby controlled, and quantification may be achieved by comparison with the internal standard [1,8]. Although highly accurate, competitive PCR can be cumbersome and labor intensive. Because serial dilutions of the internal standard must be amplified for each RNA sample, quantification of many samples in a single experiment is difficult or impractical. Therefore, we have modified competitive PCR methodology to generate a new approach (RC-PCR) that allows for the determination of specific mRNAs in a large number of small tissue samples. Experimental accuracy was confirmed by comparing the results from competitive RT-PCR and RC-PCR; both techniques gave similar results. In addition, multiple measurements from a single sample were performed with excellent reproducibility. It is important to note that variability in RNA purity, RNA integrity, and reverse transcriptase yield and uniformity is not controlled for in these assays. This is only a minor concern, since the amplifIed products are small. For most investigators, interests center on quantifying relative differences
Clinical Chernistiy
Table 2. Relative expression of PKC a and PKC
CmRNA
42, No. 2, 1996
231
in colonlc mucosa measured
by competitive
PKC a expression Competitive
PKC
PCR
Competitive
Amount, fg/ng
Relative expressiont’
RC-PCR
1 2 3
5 2.5 26
4
15
5 6 7 8
12 8 5 11
1.00 0.50 5.20 3.00 2.40 1.60 1.00 2.20
1.00 0.44 5.46 2.94 2.26 1.52 0.88 2.22
Sample
Data
are expressed as means from two separate PKC a or per ng of total RNA.
a
fg of
‘
Relative expression
was calculated
Amount,
by assigning
an arbitrary
PKC a expression Sample
Relative expressiont’
2 3 RNA from
a
each
three
colonic
and reverse-transcribed.
PKC CV,
1.00 ± 0.01 0.44 ± 0.01 5.46 ± 0.09
%
sample
a value of
mucosa
samples
Subsequently,
aliquoted
cDNA5 from each
%
10.3 tube
Ing for critical
reading
by NTH grant
gene
Nature 1988:333:859-60. G. Perrin, S. Blanckard K, Bunn HF. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Nati Acad Sci U S A 1990:87:
were
of this manuscript. CA59034.
index.
3. Gilliland
tubes
In summary, we have developed methodology that specifically, sensitively, and rapidly measures relative gene expression. RCPCR is technically straightforward, eliminating the need for radioactive isotopes, blotting, hybridization, and autoradiography. This method requires only a small amount of partially purified total RNA (0.5-1 g) and allows the relative measurement of a large number of samples, which could prove useful for both experimental and clinical studies.
Nancy
1.00 3.46 1.55 3.71 6.51 3.28 4.05 0.76
2. Chelly i, Kaplan JC, Maire P, Gautron 5, Kahn A. Transcription of the dystrophin gene in human muscle and non-muscle tissue.
10.9
between control and treatment groups. However, competitive cRNA standards may be generated to determine absolute amounts if desired [1].
e thank
RC-PCR
1.00 3.75 1.67 3.75 6.67 3.33 4.17 0.83
References
aliquoted into four separate tubes each and PKC and a expression determined by Rc-PcR. All values are means ± SE. Data are from four separate experiments. b Refer to Table 2 for details.
his work was supported
Relative expressiont’
WC, Adamovicz J. The use of the PCR to quantitate expression. PCR Methods AppI 1994;3:s123-35.
5.7
into eight
a relative
1. Gause
CV,
1.00 ± 0.03 3.46 ± 0.19 1.55 ± 0.08 were
1 and establishing
expression
Relative expressiont’
2.2 5.1 3.4
12 45 20 45 80 40 50 10
PCR
experiments.
Table 3. Assessmentof RC-PCR reproducibillty.a
1
fg/ng”
RT-PCR and RC-PCR.
expression
2725-9.
4. Ballagi-Pordany A, Ballagi-Pordany A, Funa K. Quantitative determination of mRNA phenotypes by the polymerase chain reaction. Anal Biochem 1991:196:89-94. 5. Davidson LA, Jiang YH, Derr JN, Aukema HM, Lupton JR. Chapkin RS. Protein kinase C isoforms in human and rat colonic mucosa. Arch Biochem Biophys 1994:312:547-53.
6.
Forster E. Rapid generation
of internal standards for competitive by low-stringency primer annealing. Biotechniques 1994;16: 18-20. 7. un CF. Mata M, Fink Di. Rapid construction of deleted DNA fragments for use as internal standards in competitive PCR. PCR Methods AppI 1994:3:252-5. PCR
8. Ferre F. Quantitative or semi-quantitative PCR: reality versus myth. PCR Methods AppI 1992:2:1-9. 9. Gilliland G, Perrin S, Bunn HF. PCR protocols: a guide to methods and applications. In: Innis MA, Gelfand DH, Sninsky ii, White Ti, eds. San Diego: Academic Press, 1990:60-9. 10. Clementi M, Menzo 5, Bagnarelli P, Manzin A, Van eza A, Varaldo PE. Quantitative PCR. RT-PCR in virology. PCR Methods AppI 1993:2:191-6. ii.. Piatak M Jr, Saag MS. Yang LC, Clark Si, Kappea IC, Luk KC, et al. High levels of HIV-1 in plasma during all stages of infection determined by competitive PCR. Science 1993:259:1749-54.
1
Molecular Pathology
(1996)
and Genetks
Rapid competitive PCR determination of relative gene expression in limiting tissue samples YL-HAL
JIANG,
LAURIE
A.
DAVIDSON,
JOANNE
Reverse transcriptase (R1)-PCR is widely used to study gene transcription in many biological systems. Despite the development of a variety of procedures, quantification of RT-PCR products remains difficult, particularly when pro-
sis, agarose
TERMS:
reverse
#{149} protein
transcriptase
kinase
#{149} mRNA.
and
LUPTON,
ROBERT
S.
CHAPKLN*
series of sensitive applications for quantification of specific mRNAs in biological samples [2-4J. Quantitative PCR is rapidly becoming a common procedure because of its high sensitivity and relative speed compared with Northern blot hybridization or RNase protection assays /1]. Most PCR applications for examining gene expression can be divided into two categories, competitive and noncompetitive. Competitive reverse transcriptase (RT)-PCR, the most widely used and well-established method, involves same-tube coamplification of the gene of interest and a synthetic internal standard fragment [3]I It is necessary, in competitive RT-PCR, to perform a series of amplifications with different amounts of target and competitor. Therefore, it is not suited for routine use, especially for processing a large number of tissue samples, since it requires several reaction tubes for each unknown sample, increasing the requirement for total RNA as well as time and reagents. In noncompetitive RT-PCR, it is necessary to normalize with an internal reference gene, such as actin or glyceraldehyde-3-phosphate dehydrogenase. Therefore, a second competitive PCR must be performed for each sample [2, 4]. Alternatively, quantification of PCR products by isotope incorporation or Southern hybridization with radioactive probes is inconvenient. In all, these tedious methods are useful for processing a small number of samples. In our studies on the relation between gene expression of protein kinase C (PKC) isoenzymes and colon tumorigenesis, we developed the more rapid reverse competitive (RC)-PCR approach for the analysis of a large number of very limiting colonic mucosal specimens. This strategy represents a modification of the competitive RT-PCR approach. Basically, relative gene expression is determined by coamplification of an exogenous DNA fragment of a different size with respect to the sample cDNA, but with identical 5’- and 3’-end sequences, so that both products are amplified with the same primers. After PCR, the amplified DNA was resolved by gel electrophoresis. The relative expression of specific genes is determined by densitometric analysis of both target and competitor hands. A major advantage of the RC-PCR procedure is that only one tube
cessing a large number of samples. Therefore, we developed a novel alternative PCR technique that we term “rapid competitive PCR” (RC-PCR), designed to study the relative expression of specific genes in a large number of small tissue biopsies. RC-PCR is characterized by measuring relative gene expression at the mRNA level of two or more samples with a nonradioactive assay based on competitive PCR amplification between identical sequences of internal standard and target cDNA. Only a single reaction tube per sample is used in this technique, and it was validated by comparing RC-PCR of protein kinase C and a expression in rat colonic mucosa samples with competitive RT-PCR analysis (requiring 6-8 reaction tubes per sample). We conclude that RC-PCR is a simple, rapid, highly sensitive technique that is capable of detecting less than twofold differences in mIRNA expression. INDEXING
R.
electrophore-
C
The quantitative measurement of gene expression is of major importance in addressing many fundamental questions in biology and medicine. However, in many clinical and animal studies, determination of the relative expression of specific mRNAs in a large number of small tissue samples is required to assess gene expression under physiological and pathological conditions. Northern analysis and RNase protection assays, widely used to quantify specific mRNAs, require large amounts of total RNA and are time consuming. Thus, they are not suitable for large clinical and animal studies requiring gene expression analysis [1]. The advent of PCR has resulted in the implementation of a
Faculty of Nutntion. Molecular and Cell Biology Group, l’exas A&M University, College Station, TX 77843-247!. * Address correspondence to this author at: 442 Kleherg Center, ‘Texas A&M University, College Station, TX 77843-247!. Fax 409-845-6433; e-mail [email protected]. Received September I, 1995; accepted October 30, 1995.
Nonstandard rapid competitive
227
abbreviations: PCR;
and PKC.
R1’-PCR, protein
reverse kinase
transcriptase C.
PCR;
RC-PCR,
et al.: Rapid competitive
Jiang
228
PCR
of mRNA
____
Ja
Primers
cDNA
+
1#{176}PCR
-s.b
Generation
of
Va
Composite primer
‘%
internal
C
standard
__________________ Competitor (internal
I
I
standard, 584 bp)
b
__,__.._.*.*#iInternal
B
CDNA
cI)NA, equal amounts
IS
Serial dilutions of IS
4,
RC-PCR
PCR and Electrophoresis
and RT.PCR
680 bp
PCR and Electrophoresis
680 584
bp
Competitive
RC-PCR
Fig. 1. Schematic
view of internal standard
generation
with subsequent
RC-PCR and competitive
RT-PCR
RT-PCR.
(A) Generation of internal standard. The black regions on the cDNA are complementary
to PcR primers b and c. To produce a smaller intemal standard (competitor), primer a contains primer cat its 5 end and a 13-bp region at its 3 end, which is designed PCR, primers b and c are used to generate a large quantity of the internal standard. (B)
primer band composite primer a were used during 1* PCR. The composite complementary region of the eDNA. During 2* Quantification of relative PKC gene expression by using RC-PCR and competitive RT.PCR.For RC-PCR,one tube containing cDNA and internal standard (IS) is amplified, electrophoresed. stained, and scanned for quantification. For competitive RT-PcR, seven tubes containing equal amounts of eDNA and dilutions of internal standard are amplified, electrophoresed, stained, and the equivalence estimated by eye. 680 bp: targeted PKC gene; 584 bp: internal standard (competitor). to anneal to an internal
is required for coamplification of the gene of interest and internal standard. For the purpose of validation of RC-PCR methodology, competitive RT-PCR was performed simultaneously to measure the relative expression of PKC and a mRNA in eight different rat colonic mucosal samples. Both methodologies gave similar results.
Materials and Methods RNA
EXTRACTION
AND
REVERSE
TRANSCRIPTION
Total RNA was extracted from rat colonic mucosa by using Totally RNA’ as per the manufacturer’s instructions (Ambion, Austin, TX). First-strand cDNA was synthesized in a 20-/.LL total volume by using oligo(dT) primer and SuperScript II reverse transcriptase (Gibco BRL, Gaithersburg, MD) as we have previously described [5].Briefly, rat colon total RNA (1 j.Lg) and 300 ng of oligo(dT) primer were denatured by heating to 70 #{176}C for 10 mm, quickly chilled on ice, and subsequently reverse-transcribed by incubation with 0.01 mol/L dithiothreitol, 1 mmol/L dNTPs, and 200 U of SuperScript II reverse transcriptase for 1 h at 37 #{176}C, followed by enzyme denaturation at 95 #{176}C for 5 mm. Incubations containing no reverse transcriptase were used as negative controls. COMPETITIVE
RT-PCR
For validation purposes, competitive RT-PCR was performed. A schematic view of internal standard generation is shown in
Fig. 1. A single set of primers to amplify both target cDNA and an added competitor (internal standard) of known concentration was used. Internal standards for PKC and a were constructed as previously described [6, 7]. The primer pairs used for amplification of PKC and a are listed in Table 1. For each sample, 1 jg of total RNA was routinely used for cDNA synthesis. PCR was conducted with a GeneAmp PCR System 2400 (PerkinElmer, Foster City, CA) in a total volume of 50 .tL containing 1 .tL (0-100 ng) of cDNA template and 1 .tL of internal standard (156.2 5-10 000 fg), 20 pmol of each primer, 500
Table 1. PrImer pairs
for constructionof internal standards of PKC Cand a mRNA.
and amplification
PKC competitor (584 bp) Forward: 5’-CGATGGGGTGGATGGGATCAAAA-3’ Reverse: 5’-GTATrCATGTCAGGI3TTGTCTGGAmCGGGGGCG-3’ PKC mRNA (680 bp) Forward: 5’-CGATGGGGTGGATGGGATCAAAA-3’ Reverse: 5’-GTAUCATGTCAGGG11GTCTG-3’ PKC a competitor (293 bp) Forward: 5’-TGAACCCTCAGTGGA.ATGAGT-3’ Reverse: 5’-GGCTGCTrCCTGTCTTCTGAACTTGGCmCTCGAAC-3’ PKC a mRNA (325 bp) Forward: 5’-TGAACCCTCAGTGGAATGAGT-3’ Reverse: 5-GGCTGC1TCCTGTCTrCTGAA-3’
Clinical Chemistiy 42, No. 2, 1996
jimollL dNTPs, 10 mmol/L Tris-HCI (pH 9.0), 50 mmol/L KC1, 1.5 mmol/L MgCI,, and 1.25 U of Taq DNA polymerase (Gibco BRL). To minimize tube-to-tube differences, a master amplification mixture was prepared for every assay. An amplification program (28 cycles) of denaniration (93 #{176}C, 15 s), annealing (59 #{176}C, 15 s), and extension (74 #{176}C, 45 s) was used, followed by incubation at 74 #{176}C for 7 mm. All reactions were initially denatured at 93 #{176}C for 1 mm before PCR amplification. After completion of PCR, 10-g.tL aliquots of the reaction mixtures were analyzed on 4% NuSieve agarose gels (FMC Bioproducts, Rockland, ME), followed by staining in ethidium bromide. Sample mRNA was quantified by titrating a known quantity of the internal standard against a constant amount of the sample cDNA target. The concentration at which the product from the standard target equalled the product from the experimental target was taken to be the concentration of the sample mRNA. The relative amount of sample (target) mRNA was calculated by assigning an arbitrary sample a value of 1 and establishing a relative index to allow for comparison with RC-PCR results described below. RELATIVE
EXPRESSION
OF SPECIFIC
MRNA
MEASURED
BY
RC-PCR
RC-PCR, reverse transcription and PCR were performed as described above, with the following modifications. For each sample, only one PCR tube was used instead of seven tubes in competitive RT-PCR (Fig. 1). All samples were supplemented with an identical amount of competitor, and PCR products were For
separated
gel electrophoresis. A range of RNA numbers, and the ratio between internal standard and sample were determined to ensure that amplification was quantitative. Band intensities of photographed ethidium bromide-stained gels were determined with a BioImage Visage 1 lO-2D imaging instrument (Kodak, Ann Arbor, MI) to quantify colonic PKC and a gene expression. The relative amount of sample mRNA was calculated by assigning an arbitrary sample a value of 1 and establishing a relative index. different
OF INTERNAL
To eliminate during DNA used
as
STANDARDS
the confounding effect of variable PCR efficiency amplification, we generated DNA fragments that internal standards for both competitive PCR and
RC-PCR. The procedure used to produce internal standard DNA fragments was described previously [6, 7]. Briefly, a composite primer listed in Table 1 was used in PCR reactions to generate a slightly smaller product, which was confirmed by restriction enzyme digestion. This DNA fragment was subsequently purified, quantified by ultraviolet absorption, and used in both competitive RT-PCR and RC-PCR for the measurement of the relative expression of PKC and a rnRNAs in colonic mucosa. RC-PCR
CONDITIONS
establish
expression optimal
I-
0 Si
C.
E 0 0
I-
C is
is C ISi
C
Si I-
is
I-
0
40
20
60
80
100
120
RNA (ng) Fig. 2. (A) Electrophoresis of products from rat colon PKC RC-PCR; (B) linear plot of data obtained by densitometric scanning of (A). (A) Total RNA(1 sag)was reverse-transcribed into cDNA as described in Materials and Methods, and twofold dilutions of rat colonic total RNA (lanes, from left to right:0, 3.125, 6.25, 12.5, 25, 50, 100 ng) were mixed with a constant amount (1 pg) of internal standard DNA (PKC competitor). PCR products were separated with a 4% NuSieve agarose gel, followed by ethidium bromide (5 mg/L) staining. PRC, rat PKC pcR product band (680 bp); IS, internal standard (584 bp). (B) The ratio (target/competitor) from densitometry of the gel shown in (A) was plotted against the amount of cDNA.Regression line slope = 0.08 (R2 = 0.96).
cycle
Results PREPARATION
To
A
by agarose
amounts,
were
229
of
PCR
AND
ASSAY
RC-PCR
for
specific
genes,
conditions.
the Once
it
LIMITS
measurement of the relative was necessary to determine the reaction conditions were
established and optilnized, the appropriate starting amount of sample RNA was empirically determined to ensure that a constant ratio between the target and the internal standard existed. For the purpose of determining the effect of sample mass (initial PCR reaction template) on the ratio of amplified sample to internal standard, RNA (1 ig) was reverse-transcribed to generate cDNA. Subsequently, a series of twofold dilutions of the cDNA (equivalent to 1.5625-100 ng of starting RNA) was coamplifled with a constant amount of PKC internal standard at the optimal number of cycles (28 cycles) to determine the linear reaction range (Fig. 2). The linear amplification range for PKC and a was from 10-100 ng and 5-50 ng of sample cDNA, respectively. To examine the effect of cycle number on PKC mRNA expression in rat colon, cDNA from 40 ng of total RNA and a constant amount of internal standard were coamnplified by RC-PCR over a range of amplification cycles. The proportionality of both target and standard mass remained constant from 21 to 32 cycles (Fig. 3). COMPARISON
To validate
OF RC-PCR
RC-PCR
AND
COMPETITIVE
methodology,
RT-PCR
we compared
results
(rela-
tive gene expression) with those obtained by competitive RTPCR. Total RNA preparations from rat colon were subjected to competitive RT-PCR with increasing amounts of internal standard (competitor DNA) (Fig. 4), and the absolute amount and relative expression of mRNA were calculated (Table 2). The relative expression of PKC and PKC a in colonic samples
Jiang
230
et al.: Rapid
competitive
PCR
of mRNA
total RNA samples were divided into eight aliquots each and individually reverse-transcribed. Subsequently, four aliquots of each reverse transcriptase reaction were utilized for both PKC ,T and a RC-PCR assays. The results of the assays are shown in Table 3. The standard error and covariance (CV) were consistently low, indicating that the reverse transcriptase and PCR components of the assay are highly reproducible.
A
1.5
Discussion 19
21
23
25
27
29
31
33
35
Cycle Number
Fig. 3.
(A) RC-PCR cycle titration of colonic PKC ; (B)plot of data obtained by densitometric scanning of (A). (A) Total RNA(1 extracted from rat colonic mucosa was reverse-transcribed. Equal amounts subsequently
of cDNA from an aliquot corresponding to 20 ng of total RNA was amplified in the presence of a constant amount of internal standard (1 pg) for 19-34 cycles (lanes, from left to right: 19, 21, 24. 26, 28, 30. 32. 34 cycles). PCR products (10 L) were separated with a 4% NuSieve agarose gel, followed by ethidium bromide (5 mg/L) staining. PK , rat PKC PCRproduct band (680 bp); IS, internal standard (PKC competitor, 584 bp). (B) The ratio (target/competitor) from densitometry of gel shown in (A) was plotted
against cycle number. determined by RC-PCR was very similar to competitive RTPCR data, as shown in Table 2. To evaluate the reproducibility of the assay and variability of the reverse transcription step, three different rat colonic mucosa
I......Is
c-I
C Si C.
E 0
c-I is
C
is Si
C
0
2000
4000
6000
fg Competitor
8000
10000
12000
DNA
Fig. 4. (A) Competitive RT-PCR of colonic PKC ; (B) linear plot of data obtained by densitometric scanning of (A). (A) Total RNA(1 p.g)extracted from rat colonic mucosa was reverse-transcribed and amplified in the presence of different amounts of competitor DNA (lanes, from
left to right: 10 000,
5000, 2500, 1250, 625, 312.5, 156.25 fg). PcR (10 L) were separated with a 4% NuSieve agarose gel, followed by ethidium bromide (5 mg/L) staining. PKc , rat PKc PCR product band (680 bp); IS, internal standard (584 bp). (B) The ratio of the internal standard to the sample allows for the quantification of PKC mRNA. At the theoretical equivalence point (ratio = 1), the amount of PKC competitor DNA (internal standard) equals the amount of PKC cDNA present in the analyzed sample.
products
We report the development and validation of a simple, nonradioactive approach (RC-PCR) to determine the relative expression of specific genes. This rapid and highly reproducible technique exploits the tremendous sensitivity of competitive PCR, and has several advantages over competitive RT-PCR: Numnerous samples can be processed by RC-PCR in the same run; less total RNA is required; and the technique is much less labor intensive, costly, and time-consuming. With conventional PCR, small variations in the starting concentration of the target sequence and the efficiency of Taq DNA polymerase at each cycle can result in very large variations in the final yield of PCR product [8, 9]. Thus, quantitative comparisons between tubes are invalid because the amount of final product does not accurately reflect the amount of starting template. This is particularly true when the number of cycles is excessive and amplification efficiency decreases dramatically (plateau effect). To address these pitfalls, several approaches have been developed for quantitative RT-PCR, including (a) cycle titration, (b) serial dilutions, (c) coamplification of an unrelated endogenous mRNA for normalization of the signal, and (d) competitive PCR. The latter method [8, 10, 11] is the most rigorous and is accomplished by introducing into the sample a known amount of an exogeneous, mutated artificial internal standard that may be coamplifled in the same tube with the same primers as the mRNA of interest. The two products that are amplified may be distinguished from one another by size, restriction endonuclease cleavage, or hybridization with separate probes. Since the internal standard is coamplified with the same primers as the target sequence, the efficiency of amplification is the same as that for the endogenous mRNA. Tube-to-tube variation is thereby controlled, and quantification may be achieved by comparison with the internal standard [1,8]. Although highly accurate, competitive PCR can be cumbersome and labor intensive. Because serial dilutions of the internal standard must be amplified for each RNA sample, quantification of many samples in a single experiment is difficult or impractical. Therefore, we have modified competitive PCR methodology to generate a new approach (RC-PCR) that allows for the determination of specific mRNAs in a large number of small tissue samples. Experimental accuracy was confirmed by comparing the results from competitive RT-PCR and RC-PCR; both techniques gave similar results. In addition, multiple measurements from a single sample were performed with excellent reproducibility. It is important to note that variability in RNA purity, RNA integrity, and reverse transcriptase yield and uniformity is not controlled for in these assays. This is only a minor concern, since the amplifIed products are small. For most investigators, interests center on quantifying relative differences
Clinical Chernistiy
Table 2. Relative expression of PKC a and PKC
CmRNA
42, No. 2, 1996
231
in colonlc mucosa measured
by competitive
PKC a expression Competitive
PKC
PCR
Competitive
Amount, fg/ng
Relative expressiont’
RC-PCR
1 2 3
5 2.5 26
4
15
5 6 7 8
12 8 5 11
1.00 0.50 5.20 3.00 2.40 1.60 1.00 2.20
1.00 0.44 5.46 2.94 2.26 1.52 0.88 2.22
Sample
Data
are expressed as means from two separate PKC a or per ng of total RNA.
a
fg of
‘
Relative expression
was calculated
Amount,
by assigning
an arbitrary
PKC a expression Sample
Relative expressiont’
2 3 RNA from
a
each
three
colonic
and reverse-transcribed.
PKC CV,
1.00 ± 0.01 0.44 ± 0.01 5.46 ± 0.09
%
sample
a value of
mucosa
samples
Subsequently,
aliquoted
cDNA5 from each
%
10.3 tube
Ing for critical
reading
by NTH grant
gene
Nature 1988:333:859-60. G. Perrin, S. Blanckard K, Bunn HF. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Nati Acad Sci U S A 1990:87:
were
of this manuscript. CA59034.
index.
3. Gilliland
tubes
In summary, we have developed methodology that specifically, sensitively, and rapidly measures relative gene expression. RCPCR is technically straightforward, eliminating the need for radioactive isotopes, blotting, hybridization, and autoradiography. This method requires only a small amount of partially purified total RNA (0.5-1 g) and allows the relative measurement of a large number of samples, which could prove useful for both experimental and clinical studies.
Nancy
1.00 3.46 1.55 3.71 6.51 3.28 4.05 0.76
2. Chelly i, Kaplan JC, Maire P, Gautron 5, Kahn A. Transcription of the dystrophin gene in human muscle and non-muscle tissue.
10.9
between control and treatment groups. However, competitive cRNA standards may be generated to determine absolute amounts if desired [1].
e thank
RC-PCR
1.00 3.75 1.67 3.75 6.67 3.33 4.17 0.83
References
aliquoted into four separate tubes each and PKC and a expression determined by Rc-PcR. All values are means ± SE. Data are from four separate experiments. b Refer to Table 2 for details.
his work was supported
Relative expressiont’
WC, Adamovicz J. The use of the PCR to quantitate expression. PCR Methods AppI 1994;3:s123-35.
5.7
into eight
a relative
1. Gause
CV,
1.00 ± 0.03 3.46 ± 0.19 1.55 ± 0.08 were
1 and establishing
expression
Relative expressiont’
2.2 5.1 3.4
12 45 20 45 80 40 50 10
PCR
experiments.
Table 3. Assessmentof RC-PCR reproducibillty.a
1
fg/ng”
RT-PCR and RC-PCR.
expression
2725-9.
4. Ballagi-Pordany A, Ballagi-Pordany A, Funa K. Quantitative determination of mRNA phenotypes by the polymerase chain reaction. Anal Biochem 1991:196:89-94. 5. Davidson LA, Jiang YH, Derr JN, Aukema HM, Lupton JR. Chapkin RS. Protein kinase C isoforms in human and rat colonic mucosa. Arch Biochem Biophys 1994:312:547-53.
6.
Forster E. Rapid generation
of internal standards for competitive by low-stringency primer annealing. Biotechniques 1994;16: 18-20. 7. un CF. Mata M, Fink Di. Rapid construction of deleted DNA fragments for use as internal standards in competitive PCR. PCR Methods AppI 1994:3:252-5. PCR
8. Ferre F. Quantitative or semi-quantitative PCR: reality versus myth. PCR Methods AppI 1992:2:1-9. 9. Gilliland G, Perrin S, Bunn HF. PCR protocols: a guide to methods and applications. In: Innis MA, Gelfand DH, Sninsky ii, White Ti, eds. San Diego: Academic Press, 1990:60-9. 10. Clementi M, Menzo 5, Bagnarelli P, Manzin A, Van eza A, Varaldo PE. Quantitative PCR. RT-PCR in virology. PCR Methods AppI 1993:2:191-6. ii.. Piatak M Jr, Saag MS. Yang LC, Clark Si, Kappea IC, Luk KC, et al. High levels of HIV-1 in plasma during all stages of infection determined by competitive PCR. Science 1993:259:1749-54.
